The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase.
The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate (alpha,beta-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of alpha,beta-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in alpha,beta-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and alpha,beta-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.
Vertessy
B G
BG
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest. vertessy@enzim.hu
Larsson
G
G
Persson
T
T
Bergman
A C
AC
Persson
R
R
Nyman
P O
PO
D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
C110398:2'-deoxyuridine 5'-imidotriphosphate; D003856:Deoxyuracil Nucleotides; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D008274:Magnesium